In this study, we first compared relative telomere length and TERT expression in GC, AMH levels in follicular fluid between elderly patients and young patients in IVF/ICSI treatment. We found that there were significant differences between these three biomarkers with regard to age. Correlations between relative telomere length, relative TERT expression, follicular fluid AMH levels and the ovarian/embryonic outcome, probability of clinical pregnancy were also assessed.
The current study demonstrates that TERT in GC and AMH levels in follicular fluid predicts ovarian response better than RTL during IVF treatment. However, all of them have no potential predictors of embryonic development for infertile women undergoing fertility treatment. In addition, the present study also indicates that FF AMH is associated with basal serum FSH. Finally, The ROC analyses indicated that higher FF AMH levels may predict a higher probability of clinical pregnancy in advanced women.
It is well established that TL and telomerase activity are known biomarkers for biological aging [29]. Low telomerase activity is associated with unexplained ovarian dysfunction in women [30], and a positive correlation is found between the success of IVF treatment and telomerase activity in granulosa cells [10, 31]. TERT encodes the catalytic subunit of telomerase, thereby counters telomere shortening during cell division [32].The TERT mRNA codifies for the catalytic component of telomerase, with the other enzyme component TERC which is a RNA template [33]. Both components constitute active telomerase, which elongates the gradual shortening of telomere with each round of DNA replication by maintaining the telomere sequences and conferring sustained proliferation capacity to growing cells [34]. GC cells supporting the oocytes during follicular growth and maturation, exploring knowledge about aging of the GC gives us important insights into oocyte aging [35]. Telomerase activity in GC is important for their proliferation and differentiation capacity [36], as it plays versatile roles in various reproduction pathways. Therefore, we select GC cells which are typically discarded during IVF treatment as research samples.
Here, we analysed the correlation between the expression level of major telomerase gene TERT and telomere length in ovarian and embryonic outcome. The results reveals that the relative telomere length of GC samples in the present study is not associated with any measure of ovarian or embryonic performance but age, which was not in accordance with the previous report that GC relative telomere length does not change significantly with aging [11]. Rather, TERT is associated with age and ovarian response but not embryonic performance. This finding stands in contrast to a number of previous studies that have demonstrated variations in embryonic outcomes in the setting of shorter or longer GC telomere length [37, 38]. All these findings indicate that, TERT appears to be a more significant biomarker for predicting the ovarian response among infertile women, both TERT and RTL have no role in predicting embryonic development potential as no statistically correlation is noted between TERT/RTL and blastocyst formation rate. Findings of some researchers support the decline in TERT expression and activity in mouse oocytes with reproductive aging [34] and its association with telomere shortening and decline in oocyte quality [39]. Interestingly, our data indicated an increasing in TERT expression in GC with aging and our unpublished RNA-sequencing data showed no significant changes of TERT expression between old and young human oocytes. These seemingly discordance results demonstrate that the regulation manner of TERT expression is tissue-dependent [13].
In addition to its established role of compensating for cell replication-dependent telomere shortening, TERT exhibits multiple physiological activities beyond its canonical action. For instance, TERT is shown to modulate mitochondrial and ubiquitin proteasomal function or act as a transcription co-factor [40], to regulate gene expression, to promote cell survival and growth, to protect cells from apoptosis [32], and to display the RNA-dependent RNA polymerase activity [40].A pleiotropic role for TERT in regulating the epigenetic clock [32] and intrinsic DNA methylation age during cell proliferation on advancing cells are also indicated [29].Together, all these TERT effects are actively involved in biological activities, ultimately affecting aging process. In the present study, relative telomere length became shorter while TERT mRNA expression increased with aging in GC. TERT possessing activities dependent or independent on telomere maintenance may contribute to this aging process.
GC cells produce the female hormones. Telomerase activity in GC is under the control of growth factors and steroid hormones [36]. The microenvironment of granulosa cell contains oestrogens reported to influence telomerase activity and telomere length [35, 41]. Enhancement of TERT expression may increase the steroidogenic genes expression which is important for various physiological functions such as reproduction. These confer the importance of endocrine in ovarian microenvironment. Although the telomere theory of reproductive aging has mostly focused on the effect of shorter telomeres on meiotic errors and higher risk of aneuploidies [29, 41], it is also possible that the length of telomeres or telomerase interaction with endocrine in ovarian microenvironment may play a role in ovarian reserve.
AMH is produced by ovarian granulosa cells, primarily in pre-antral and small antral follicles less than 8 mm in diameter [24]. It plays a pivotal role in folliculogenesis [35]. The expression of AMH [both mRNA and protein] in human granulosa cells wanes with increasing size of the human follicles [42–43], reaching a stable low level in GC isolated from pre-ovulatory follicles before ovulation induction and at oocyte retrieval [44]. Currently, serum AMH has been widely used as a golden maker for evaluating ovarian reserve of females, particularly in the field of assisted reproduction [45]. and it was associated with increased cumulative ongoing pregnancy in a study [46]. However, FF AMH can also not be ignored. A prospective study investigated AMH level in the follicular fluid and mRNA expression pattern in granulosa cells in relation to age, and an increase in FF AMH level in the older than in the younger age group was found [47]. This result was in contrast with our findings that FF AMH levels was lower from the older age group than from younger group. Whatever, these suggest that FF AMH may be involved in follicular aging process. Sacha et al. found higher FF AMH appears to be associated with a significantly higher probability of clinical pregnancy,but in their study, the patients were not grouped by age [24]. In our study, patients were grouped by age and ROC curve showed that predictive value of follicular fluid AMH levels for probability of clinical pregnancy was significant just in old age group.
Some researchers investigated whether follicular fluid AMH levels were predictive of IVF outcomes but got inconsistent results [48–51]. In present study, we found FF AMH was related to ovarian response but not embryonic development.
TERT interaction with FF AMH in ovarian microenvironment may play a role in ovarian aging process. A distinctive epigenetic aging profile in human granulosa cells refers to AMH gene [35]. Some researchers found age-related changes in the AMH gene in human granulosa cells [52]. The cross-talking between TERT and FF AMH may lead to the interaction of TERT with key signaling pathways, which is deserve to investigate and discuss in future [40].
There are still some deficiencies must be referred in the research. First of all, due to the limitation of the sample size, there might be certain bias. Secondly, GC samples are pooled from all of cumulus-oocyte complex of each patient and rather than being analyzed individually. Moreover, we do not measure all tubes of follicular fluid AMH levels in each patient on the day of oocyte retrieval. Although follicular fluid AMH concentrations show little variability between pre-ovulatory follicles in a retrospective cohort study [24], analyzing relationship between individual cumulus-oocyte complex and corresponding follicular fluid AMH levels might lead to more reliable results. Last but not least, TERT measurement with qPCR in the study is at the levels of transcription, other methods of TERT assessment such as western blot can be performed at translation level so as to confirm the results.